Crystal forms of immunomodulators

ABSTRACT

The invention relates to crystalline forms of a 3-substituted 1,2,4-oxadiazole compound, including an anhydrous crystalline form, methods of their preparation, and related pharmaceutical preparations thereof. The invention also relates to preparations suitable for pharmaceutical, veterinary, and agriculturally-relevant uses.

RELATED APPLICATIONS

This application claims the benefit of International Application No.PCT/CN2017/104,485, filed on Sep. 29, 2017. The contents of thisapplication are hereby incorporated by reference in their entirety.

BACKGROUND

The immune system in mammals regulates the activation and inactivationof lymphocytes through various mechanisms during and after an immuneresponse. Among these mechanisms, there are mechanisms that specificallymodulate the immune response as and when required.

3-substituted 1,2,4-oxadiazole compounds act as immunomodulators. Thus,3-substituted 1,2,4-oxadiazole compounds can be used in the treatment ofcancer, immune disorders, immunodeficiency disorders, inflammatorydisorders, infectious diseases, and transplant rejection.

Given the therapeutic benefits associated with 3-substituted1,2,4-oxadiazole compounds, there is a need for improved compositions ofthese compounds. Further, there is a need for improved methods forpreparing and formulating 3-substituted 1,2,4-oxadiazole compounds.

SUMMARY

One aspect of the invention relates to an anhydrous crystalline compoundhaving the structure of formula (I),

Another aspect of the invention relates to methods for preparing theanhydrous crystalline compounds of formula (I).

In certain embodiments, the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, comprising an anhydrouscrystalline compound of formula (I), and one or more pharmaceuticallyacceptable excipients. In certain embodiments, the pharmaceuticalpreparations may be for use in treating or preventing a condition ordisease as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the XRPD patterns of formula (I) Form A.

FIG. 2 shows the XRPD patterns of anhydrous formula (I) Form A fromdifferent crystallization conditions.

FIG. 3A shows crystals from the wet cake formed with 5 wt % seedcrystals and addition of EtOH over 8 h using polarized light microscopy.

FIG. 3B shows crystals from the dried cake formed with 5 wt % seedcrystals and addition of EtOH over 8 h using polarized light microscopy.

FIG. 4A shows crystals from the wet cake formed with 5 wt % seedcrystals and addition of EtOH over 4 h using polarized light microscopy.

FIG. 4B shows crystals from the dried cake formed with 5 wt % seedcrystals and addition of EtOH over 4 h using polarized light microscopy.

FIG. 5A shows crystals from the wet cake formed with 1 wt % seedcrystals and addition of EtOH over 4 h using polarized light microscopy.

FIG. 5B shows crystals from the dried cake formed with 1 wt % seedcrystals and addition of EtOH over 4 h using polarized light microscopy.

FIG. 6A shows crystals from the wet cake formed with 5 wt % seedcrystals and addition of EtOH/H₂O over 4 h using polarized lightmicroscopy.

FIG. 6B shows crystals from the dried cake formed with 5 wt % seedcrystals and addition of EtOH/H₂O over 4 h using polarized lightmicroscopy.

FIG. 7 shows the thermogravimetric analysis and differential scanningcalorimetry thermogram of anhydrous Form A.

FIG. 8 shows a comparison of XRPD patterns of anhydrous formula (I) FormA initially, at 25° C., 60% relative humidity (RH); 40° C., 75% RH; and60° C. after three months compared to a Form A reference sample in solidform.

FIG. 9 shows a comparison of XRPD patterns of anhydrous formula (I) FormA in water at 20° C.; in water at 30° C.; and Form A after solubilitytesting.

FIG. 10 shows a dynamic vapor sorption isotherm of anhydrous formula (I)Form A.

FIG. 11 shows a comparison of XRPD patterns of anhydrous formula (I)Form A before and after dynamic vapor sorption.

FIG. 12A shows the asymmetric unit of the anhydrous formula (I) Form Asingle crystal.

FIG. 12B shows a proposed proton transfer to form a zwitterion in theanhydrous formula (I) Form A crystal.

FIG. 12C shows the molecular formula of anhydrous formula (I) Form A inthe single crystal.

FIG. 13 shows a comparison of XRPD patterns of anhydrous formula (I)Form A based on a sample and as calculated from the single crystalstructure.

DETAILED DESCRIPTION

In certain embodiments, the invention provides an anhydrous crystallinecompound having the structure of formula (I),

In some embodiments, the invention provides an anhydrous crystallinecompound having the structure of formula (I) showing all of the atoms,

In certain embodiments, a crystalline compound of formula (I) is notsolvated (e.g., the crystal lattice does not comprise molecules of asolvent). In certain embodiments, the crystalline compound of formula(I) is anhydrous, or substantially anhydrous.

Any crystalline compound described herein may be used in the manufactureof a medicament for the treatment of any diseases or conditionsdisclosed herein.

In certain embodiments, the compounds of the present invention canassemble into more than one crystal formation. These different forms areunderstood as “polymorphs” herein.

In certain embodiments, the polymorph of the crystalline compound ischaracterized by powder X-ray diffraction (XRD). θ represents thediffraction angle, measured in degrees. In certain embodiments, thediffractometer used in XRD measures the diffraction angle as two timesthe diffraction angle θ. Thus, in certain embodiments, the diffractionpatterns described herein refer to X-ray intensity measured againstangle 2θ.

In certain embodiments, an anhydrous crystalline compound of formula (I)has 20 values 18.8±0.2, 20.5±02, 26.4±0.2, 27.5±0.2, and 32.0±0.2. Infurther embodiments, the anhydrous crystalline compound has 2θ values18.8±0.2, 20.5±0.2, 26.1±0.2, 26.4±0.2, 27.5±0.2, 30.6±0.2, and32.0±0.2. In yet further embodiments, the anhydrous crystalline compoundhas 2θ values 15.8±0.2, 16.5±0.2, 18.8±0.2, 20.5±0.2, 25.5±0.2,26.1±0.2, 26.4±0.2, 27.5±0.2, 30.6±0.2, and 32.0±0.2. In still yetfurther embodiments, the anhydrous crystalline compound has 2θ values15.8±0.2, 16.5±0.2, 18.8±0.2, 20.5±0.2, 21.8±0.2, 22.3±0.2, 24.2±0.2,25.5±0.2, 26.1±0.2, 26.4±0.2, 27.5±0.2, 30.4±0.2, 30.6±0.2, and32.0±0.2. In some embodiments, the anhydrous crystalline compound has 2θvalues selected from the following peaks listed Table 1±0.2.

TABLE 1 Exemplary peaks of Form A FWHM Left d-spacing Rel. Int. Pos.[°2Th.] Height [cts] [°2Th.] [{acute over (Å)}] [%] 10.151280 108.2655000.153504 8.71403 3.15 15.169030 233.352500 0.102336 5.84094 6.7815.811010 766.866000 0.153504 5.60519 22.29 16.494550 716.3964000.153504 5.37442 20.82 17.600160 210.694600 0.102336 5.03923 6.1218.132870 339.663200 0.127920 4.89237 9.87 18.820550 3441.0600000.153504 4.71513 100.00 20.494370 1153.954000 0.153504 4.33366 33.5321.789780 459.031400 0.127920 4.07886 13.34 22.335970 447.6237000.153504 3.98034 13.01 22.935230 293.402500 0.153504 3.87768 8.5324.235630 427.814300 0.153504 3.67248 12.43 25.446040 679.3513000.153504 3.50047 19.74 26.095330 820.166200 0.127920 3.41483 23.8326.444450 1109.730000 0.153504 3.37053 32.25 27.454720 1759.5470000.153504 3.24876 51.13 28.071180 323.452300 0.179088 3.17880 9.4028.750540 189.132400 0.179088 3.10521 5.50 29.347520 184.759400 0.2046723.04339 5.37 30.431090 460.327500 0.127920 2.93745 13.38 30.644910576.765100 0.127920 2.91744 16.76 31.956340 1212.746000 0.179088 2.8006535.24 33.361460 285.081400 0.102336 2.68583 8.28 33.604180 270.9694000.179088 2.66699 7.87 34.606870 310.585900 0.153504 2.59198 9.0335.841170 145.972200 0.358176 2.50550 4.24 36.746890 117.362000 0.2558402.44579 3.41 37.315610 48.631210 0.204672 2.40982 1.41 38.218310211.915300 0.255840 2.35495 6.16 38.584910 122.935200 0.153504 2.333413.57 39.160630 71.010390 0.153504 2.30043 2.06

In certain embodiments, an anhydrous crystalline compound of formula (I)has an XRD pattern substantially as shown in FIG. 1 , labeled Form A.

In some embodiments, an anhydrous crystalline compound of formula (T)shows a differential scanning calorimetry (DSC) melting/decompositionwith an onset temperature in the range selected from about 197° C. toabout 210° C., about 201° C. to about 206° C., and about 202° C. toabout 205° C. In certain embodiments, a crystalline compound of formula(I) is not solvated (e.g., the crystal lattice does not comprisemolecules of a solvent).

In certain embodiments, the invention relates to a pharmaceuticalcomposition comprising an anhydrous crystalline compound of formula (I)and one or more pharmaceutically acceptable excipients. In certainembodiments, the pharmaceutical composition is selected from tablets,capsules, and suspensions.

The term “substantially pure” as used herein, refers to an anhydrouscrystalline polymorph that is greater than 90% pure, meaning that itcontains less than 10% of any other compound, including thecorresponding amorphous compound or an alternative polymorph of thecrystalline salt. Preferably, the anhydrous crystalline polymorph isgreater than 95% pure, or even greater than 98% pure.

Methods of Making the Anhydrous Crystalline Forms of the Compound ofFormula (I)

In certain embodiments, the invention relates to a method for preparingan anhydrous crystalline compound having the structure of formula (I),comprising:

a) providing a mixture comprising a compound of formula (I) and asolvent; andb) crystallizing the compound of formula (I) from the mixture comprisingthe compound of formula (I).

In certain embodiments, the mixture comprising the compound of formula(I) and the solvent is a reaction mixture.

In certain embodiments, the mixture comprising the compound of formula(I) is a solution. In certain embodiments, the solution comprises acompound of formula (I) dissolved in a solvent. In some embodiments, thesolution comprises a crude solid material comprising the compound offormula (I) dissolved in a solvent. In some embodiments, the solutioncomprises a reaction mixture.

In certain embodiments, the mixture is a slurry or a suspension. Incertain embodiments, the slurry or the suspension comprises crude solidmaterial comprising the compound of formula (I).

In certain embodiments of the solutions, slurries, and suspensionsdisclosed herein, the crude solid material comprising the compound offormula (I) is less than 70% pure, less than 75% pure, less than 80%pure, less than 85% pure, or less than 90% pure with respect to thecompound of formula (I). In certain embodiments, the crude solidmaterial comprising the compound of formula (I) is less than 90% purewith respect to the compound of formula (I). In certain embodiments, thecrude solid material comprises about 70% to about 90% compound offormula (I). In some embodiments, the purity of the crude solid materialis about 70% to about 90% with respect to the compound of formula (I).

In certain embodiments, after crystallization, the compound of formula(I) is substantially pure. In some embodiments, the anhydrouscrystalline form of the compound of formula (I) is greater than 90%pure. In some embodiments, the purity of the anhydrous crystalline formof the compound of formula (I) is selected from greater than 90%,greater than 91%, greater than 92%, greater than 93%, greater than 94%,greater than 95%, greater than 96%, greater than 97%, greater than 98%,and greater than 99%. In some embodiments, the purity of the anhydrouscrystalline form of the compound of formula (I) is greater than 95%. Insome embodiments, the purity of the anhydrous crystalline form of thecompound of formula (I) is greater than 98%. In some embodiments, thepurity of the anhydrous crystalline form of the compound of formula (I)is selected from about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, and about 99%.

In certain embodiments, the crystalline compound made by the methods ofthe invention is anhydrous. In certain embodiments, provided herein is amethod for preparing an anhydrous crystalline compound having thestructure of formula (I):

comprising:a) providing a mixture comprising a compound of formula (I) and asolvent; andb) crystallizing the compound of formula (I) from the mixture comprisingthe compound of formula (I).

In some embodiments, solvent vapor slowly diffuses into a solid sample.

In certain embodiments, the mixture comprising the compound of formula(I) is a solution, and the step of crystallizing the compound from themixture comprises bringing the solution to supersaturation to cause thecompound of formula (I) to precipitate out of solution.

In certain embodiments, bringing the mixture comprising the compound offormula (I) to supersaturation comprises the slow addition of ananti-solvent, such as heptanes, hexanes, ethanol, or another polar ornon-polar liquid miscible with an aqueous solution, allowing thesolution to cool (with or without seeding the solution), reducing thevolume of the solution, or any combination thereof. In certainembodiments, the anti-solvent is ethanol. In certain embodiments,bringing the mixture comprising the compound of formula (I) tosupersaturation comprises adding an anti-solvent, cooling the solutionto ambient temperature or lower, and reducing the volume of thesolution, e.g., by evaporating solvent from the solution. In certainembodiments, allowing the solution to cool may be passive (e.g.,allowing the solution to stand at ambient temperature) or active (e.g.,cooling the solution in an ice bath or freezer).

In certain embodiments, bringing the mixture comprising the compound offormula (I) to supersaturation comprises adding an anti-solvent,maintaining a solution temperature at ambient temperature or higher, andreducing the volume of the solution, e.g., by evaporating solvent fromthe solution. In some embodiments, the step of bringing the solution tosupersaturation comprises maintaining a solution temperature at ambienttemperature or higher. In some embodiments, the step of bringing thesolution to supersaturation comprises maintaining a solution temperatureabove about 20° C. In some embodiments, the step of bringing thesolution to supersaturation comprises maintaining a solution temperatureat about 20° C., about 25° C., about 30° C., about 35° C., about 40° C.,about 45° C., about 50° C., about 55° C., about 60° C., about 65° C., orabout 70° C.

In certain embodiments, the preparation method further comprisesisolating the crystals, e.g., by filtering the crystals, by decantingfluid from the crystals, or by any other suitable separation technique.In further embodiments, the preparation method further comprises washingthe crystals.

In certain embodiments, the preparation method further comprisesinducing crystallization. The method can also comprise drying thecrystals, for example under reduced pressure. In certain embodiments,inducing precipitation or crystallization comprises secondarynucleation, wherein nucleation occurs in the presence of seed crystalsor interactions with the environment (crystallizer walls, stirringimpellers, sonication, etc.).

In other embodiments, the solvent is acetonitrile, diethyl ether,N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulfoxide(DMSO), dichloromethane, ethanol, ethyl acetate, heptanes, hexanes,isopropyl acetate, methanol, methylethyl ketone, N-methyl-2-pyrrolidone(NMP), tetrahydrofuran, toluene, 2-propanol (isopropanol), 1-butanol,water, or any combination thereof. In some embodiments, the solvent isdichloromethane. In some embodiments, the solvent is tetrahydrofuran.

In some embodiments, the anti-solvent is selected from tetrahydrofuran,methanol, isopropanol, diethyl ether, ethanol, 1,4-dioxane,acetonitrile, and acetone. In some embodiments, to an aqueous solutioncomprising the compound of formula (I) is slowly added an anti-solvent.

In some embodiments, the anti-solvent is selected from tetrahydrofuran,methanol, isopropanol, 1,4-dioxane, acetonitrile, and acetone. In someembodiments, an anti-solvent slowly diffuses into an aqueous solutioncomprising the compound of formula (I).

In some embodiments, a slurry comprising the compound of formula (I) anda solvent were mixed before isolating the solids. In some embodiments,the isolation of the solids is by filtration or centrifugation.

In some embodiments, the slurry is maintained at ambient temperature orhigher. In some embodiments, the slurry is maintained at a temperatureabove about 20° C. In some embodiments, the slurry is maintained at atemperature of about 20° C., about 25° C., about 30° C., about 35° C.,about 40° C., about 45° C., about 50° C., about 55° C., about 60° C.,about 65° C., or about 70° C.

In certain embodiments, the solvent is a mixture comprising water. Insome preferred embodiments, the solvent is a mixture comprising waterand ethanol, isopropanol, methanol, or tetrahydrofuran. In certainpreferred embodiments, for example to achieve Form A, the solvent is amixture comprising water and isopropanol or water and ethanol.

In some embodiments, the solvent is a mixture comprising EtOH:H₂O in avolume-to-volume ratio selected from 19:2, 5:1, 2:1, 1:1, and 1:9. Insome embodiments, the solvent is a mixture comprising ethanol and waterto which additional ethanol or a mixture of ethanol and water is added.In some embodiments, the mixture comprises the compound of formula (I)and a solvent of 2:1 EtOH:H₂O (v/v), then a mixture of 19:2 EtOH:H₂O isadded for crystallization.

In certain embodiments, provided herein is a method for preparing ananhydrous crystalline compound having the structure of formula (I),comprising:

a) providing a mixture comprising a compound of formula (I) and asolvent;b) adding an anti-solvent to the mixture; andc) crystallizing the compound of formula (I) from the mixture comprisingthe compound of formula (I).

In some embodiments of the methods disclosed herein, the method furthercomprises adding seed crystals.

In some embodiments, crystallization is aided by seeding or seedloading, that is adding seed crystals to the mixture. In someembodiments, the seed crystals are added at a weight percentage of thetotal mixture selected from about 1 wt %, about 2 wt %, about 3 wt %,about 4 wt %, about 5 wt %, about 6 wt %, about 7 wt %, about 8 wt %,about 9 wt % a, and about 10 wt %. In some embodiments, the seedcrystals are added at a weight percentage of the total mixture selectedfrom about 1 wt %, about 2 wt %, about 3 wt %, about 4 wt %, about 5 wt%, and about 6 wt %. In some embodiments, the seed crystals are added ata weight percentage of the total mixture selected from 3 wt %, 4 wt %,and 5 wt %.

In some embodiments, the seed crystals are formula (I) Form A seedcrystals.

In some embodiments of the methods disclosed herein, the method furthercomprises adding additional anti-solvent.

In certain embodiments, provided herein is a method for preparing ananhydrous crystalline compound having the structure of formula (I),comprising:

a) providing a mixture comprising a compound of formula (I) and asolvent;b) adding an anti-solvent to the mixture;c) adding seed crystals to the mixture;d) adding additional anti-solvent to the mixture; ande) crystallizing the compound of formula (I) from the mixture comprisingthe compound of formula (I).

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, acetonitrile, ethanol, heptanes,hexanes, methanol, tetrahydrofuran, toluene, water, or a combinationthereof. As used herein, “anti-solvent” means a solvent in which thecompound crystals are insoluble, minimally soluble, or partiallysoluble. In practice, the addition of an anti-solvent to a solution inwhich the salt crystals are dissolved reduces the solubility of the saltcrystals in solution, thereby stimulating precipitation of the salt. Incertain embodiments, the crystals are washed with a combination ofanti-solvent and the organic solvent. In certain embodiments, theanti-solvent is water, while in other embodiments it is an alkanesolvent, such as hexane or pentane, or an aromatic hydrocarbon solvent,such as benzene, toluene, or xylene. In certain embodiments, theanti-solvent is methanol.

In certain embodiments, washing the crystals comprises washing theanhydrous crystalline compound of formula (I) with a solvent or amixture of one or more solvents, which are described above. In certainembodiments, the solvent or mixture of solvents is cooled prior towashing.

In certain embodiments, the methods of making the anhydrous crystallineforms of the compound of formula (I) are used to remove one or moreimpurities from the compound of formula (I). In certain embodiments, thecrystallization methods described herein are used for purifying thecompound of formula (I), e.g., as a final purification step in themanufacture of the compound.

In certain embodiments, the compound of formula (I) is purified bycrystallization. In some embodiments, purification of the compound offormula (I) does not use high-performance liquid chromatography (HPLC),including preparative HPLC. In some embodiments, purification of thecompound of formula (I) by crystallization is scalable. Advantages ofpurification by crystallization include, but are not limited to, removalof soluble impurities, ease of purification process, applicability tolarge scale synthesis, acceptable yields, and high product purity.

In some embodiments, anhydrous crystalline formula (I) Form A is stablethroughout the whole manufacturing process. In some embodiments, thewater content and physical properties of anhydrous crystalline formula(I) Form A do not change with humidity. In some embodiments, anhydrouscrystalline formula (I) Form A is not hygroscopic. For example, crystalsof anhydrous crystalline formula (I) Form A may retain their crystalstructure and not change weight by more than 0.5% when exposed to anenvironment of 75% humidity at 40° C. for a month.

In some embodiments, anhydrous crystalline formula (I) Form A was theform with greater stability. In some embodiments, the anhydrouscrystalline formula (I) Form A was formed in water or in a mixture ofethanol and water. In some embodiments, the temperature was above about10° C., above about 15° C., above about 20° C., above about 25° C.,above about 30° C., above about 35° C., above about 40° C., above about45° C., above about 50° C., above about 55° C., above about 60° C.,above about 65° C., or above about 70° C. In some embodiments, thetemperature was about 10° C., about 15° C., about 20° C., about 25° C.,about 30° C., about 35° C., about 40° C., about 45° C., about 50° C.,about 55° C., about 60° C., about 65° C., or about 70° C. In someembodiments, the temperature was about 10° C., about 15° C., about 20°C., about 25° C., or about 30° C. In some embodiments, the temperaturewas about 20° C.

In some embodiments, the conversion to anhydrous crystalline formula (I)Form A was complete in about 4 h, in about 8 h, in about 12 h, in about16 h, in about 20 h, in about 1 day, in about 2 days, in about 3 days,in about 4 days, in about 5 days, in about 6 days, in about 7 days. Insome embodiments, the conversion to anhydrous crystalline formula (I)Form A was complete in about 2 h, in about 3 h, in about 4 h, in about 5h, in about 6 h, in about 7 h, in about 8 h, in about 9 h, in about 10h, in about 11 h, in about 12 h, in about 13 h, in about 14 h, in about15 h, in about 16 h, in about 17 h, in about 18 h, in about 19 h, inabout 20 h, in about 21 h, in about 22 h, in about 23 h, in about 24 h,in about 25 h, or in about 26 h. For example, at 20° C., the conversionto anhydrous crystalline formula (I) Form A was complete in about oneday.

Uses of Anhydrous Crystal Forms of the Compound of Formula (I)

The compound of formula (I) is a 3-substituted 1,2,4-oxadiazole compoundhaving the following structure,

Functional “exhaustion” (immune dysfunction) among T and B cell subsetsis a well-described feature of chronic viral infections, such ashepatitis B and C and HIV viruses. T cell exhaustion was initiallydescribed for CD8 T cells in mice chronically infected with lymphocyticchoriomeningitis virus clone 13. In the lymphocytic choriomeningitisvirus mouse model, repeated antigen stimulation through the T cellantigen receptor drives the sustained expression of T cell inhibitoryreceptors, including programmed cell death-1 (PD-1) and lymphocyteactivationgene-3 (LAG-3), on virus-specific CD8 T cells (J. Illingworthet al., J. Immunol. 2013, 190(3): 1038-1047).

Thus, diseases modulated by an immune response including, but notlimited to, cancer, immune disorders, immunodeficiency disorders,inflammatory disorders, infectious diseases, and transplant rejection,can be treated by administering an immunomodulator, such as the compoundof formula (I), and compositions disclosed herein. 3-substituted1,2,4-oxadiazole compounds act as immunomodulators.

In certain embodiments, the compound of formula (I) modulates an immuneresponse in a cell.

In other embodiments, the present disclosure provides a method ofmodulating an immune response in a cell, comprising contacting the cellwith a composition comprising an anhydrous crystalline form of thecompound of formula (I), according to any of the above embodiments. Insome embodiments, the present disclosure provides a method of modulatingan immune response in a cell, comprising contacting the cell with acomposition comprising an anhydrous crystalline form of the compound offormula (I), according to any of the above embodiments.

In certain embodiments, the present disclosure provides uses of ananhydrous crystalline form of the compound of formula (I) for thepreparation of a medicament, e.g., for the treatment of cancer, immunedisorders, immunodeficiency disorders, inflammatory disorders,infectious diseases, and transplant rejection.

In accordance with any of the foregoing embodiments, in certainembodiments, contacting the cell occurs in a subject in need thereof,thereby treating a disease or disorder selected from cancer, immunedisorders, immunodeficiency disorders, inflammatory disorders,infectious diseases, and transplant rejection.

In certain embodiments, the present disclosure provides methods fortreating cancer, wherein the method comprises administration of atherapeutically effective amount of a composition comprising ananhydrous crystalline form of the compound of formula (I) the subject inneed thereof.

In certain embodiments, the present disclosure provides methods forinhibiting growth of tumor cells and/or metastasis by administering atherapeutically effective amount of a composition comprising ananhydrous crystalline form of the compound of formula (I) to the subjectin need thereof.

Representative tumor cells include cells of a cancer such as, but notlimited to, blastoma (e.g., glioblastoma), breast cancer (e.g., breastcarcinoma, primary ductal carcinoma, triple negative breast cancer,estrogen receptor positive (ER+), progesterone receptor positive (PR+),and/or human epidermal growth factor receptor 2 positive (HER2+)),epithelial cancer (e.g., carcinomas), colon cancer, lung cancer (e.g.,small cell lung cancer, non-small cell lung cancer (NSCLC), lungadenocarcinoma, and ling squamous cell carcinoma), melanoma (e.g.,cutaneous melanoma, ocular melanoma, cutaneous or intraocular malignantmelanoma, and lymph node-associated melanoma), prostate cancer (e.g.,prostate adenocarcinoma), renal cancer (e.g., renal cell cancer (RCC)and kidney cancer), bone cancer (e.g., osteosarcoma), pancreatic cancer(e.g., pancreatic adenocarcinoma), skin cancer, cancer of the head orneck (e.g., head and neck squamous cell carcinoma), uterine cancer,ovarian cancer (e.g., ovarian carcinoma), colorectal cancer (e.g.,microsatellite instability high colorectal cancer and colorectaladenocarcinoma), rectal cancer, cancer of the anal region, cancer of theperitoneum, stomach cancer (e.g., gastric carcinoma and gastrointestinalcancer), testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, cervical cancer (e.g., carcinoma of the cervix),vaginal cancer (e.g., carcinoma of the vagina), vulval cancer (e.g.,carcinoma of the vulva), cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, thyroid cancer (e.g., cancerof the thyroid gland), cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma (e.g., sarcoma of soft tissue and Kaposi'ssarcoma), cancer of the urethra, cancer of the penis, chronic or acuteleukemia, (e.g., acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, Hairy cellleukemia, and chronic myeloblastic leukemia), solid tumors of childhood,Hodgkin's lymphoma (HL) (e.g., lymphocyte-rich (LRCHL), nodularsclerosis (NSHL), mixed cellularity (MCHL) and lymphocyte depleted(LDHL)), B-cell lymphomas (e.g., diffuse large B-cell lymphoma (DLBCL)),non-Hodgkin's lymphoma (NHL) (e.g., low grade/follicular non-Hodgkin'slymphoma, small lymphocytic (SL) NHL, intermediate grade/follicular NHL,intermediate grade diffuse NHL, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL, bulky diseaseNHL, Burkitt's lymphoma, mantle cell lymphoma), AIDS-related lymphoma,cutaneous T-cell lymphoma (e.g., mycosis fundoides) and Waldenstrom'sMacroglobulinemia, post-transplant lymphoproliferative disorder (PTLD),lymphocytic lymphoma, primary CNS lymphoma, and T-cell lymphoma),mesothelioma, thymic carcinoma, myeloma (e.g., multiple myeloma), cancerof the bladder (e.g., bladder carcinoma), cancer of the ureter,carcinoma of the renal pelvis, liver cancer (e.g., hepatocellularcancer, hepatic carcinoma, hepatoma), pancreatic cancer, post-transplantlymphoproliferative disorder (PTLD), neoplasm of the central nervoussystem (CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, epidermoid cancer, salivary gland carcinoma, squamouscell cancer, abnormal vascular proliferation associated withphakomatoses, edema (such as that associated with brain tumors), Meigs'syndrome, Merkel cell carcinoma, environmentally induced cancers(including those induced by asbestos), and combinations of said cancers.

In other embodiments, for example, the tumor cells may include cells ofa cancer selected from prostate cancer, melanoma, breast cancer, coloncancer, prostate cancer, lung cancer, renal cancer, pancreatic cancer,gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma,thyroid cancer, thymic carcinoma, sarcoma, glioblastoma, chronic oracute leukemia, lymphoma, myeloma, Merkel cell carcinoma, epithelialcancer, colorectal cancer, vaginal cancer, cervical cancer, ovariancancer, and cancer of the head and neck.

In other embodiments, for example, the tumor cells may include cells ofa cancer selected from melanoma, triple negative breast cancer,non-small cell lung cancer, renal cell carcinoma, pancreatic cancer,gastric carcinoma, bladder cancer, mesothelioma, Hodgkins's lymphoma,cervical cancer, ovarian cancer, and head and neck squamous cellcarcinoma.

In some embodiments, the tumor cells are cells of a cancer selected fromsmall cell lung cancer, multiple myeloma, bladder carcinoma, primaryductal carcinoma, ovarian carcinoma, Hodgkin's lymphoma, gastriccarcinoma, acute myeloid leukemia, and pancreatic cancer.

In other embodiments, the tumor cells are cells of a cancer selectedfrom carcinoma of the endometrium, ovarian cancer, Hodgkin's lymphoma,non-Hodgkin's lymphoma, and chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, andmultiple myeloma.

In some embodiments, the tumor cells are cells of a cancer selected fromprostate adenocarcinoma, lung adenocarcinoma, lung squamous cellcarcinoma, pancreatic adenocarcinoma, breast cancer and colorectaladenocarcinoma. In certain embodiments, tumor cells are from breastcancer. In some embodiments, the tumor cells are from a breast cancerselected from triple negative breast cancer, estrogen receptor positive(ER+), progesterone receptor positive (PR+), and/or human epidermalgrowth factor receptor 2 (HER2+). In other embodiments, the tumor cellsare from a PAM50+ breast cancer assay panel (Parker, J. S., et al., J.Clin. Oncol., 2009, 27(8): 1160-1167), breast cancer selected fromluminal A, luminal B, HER2-enriched, basal-like and normal-like.

In some embodiments, the tumor cells are cells of a cancer selected fromtriple negative breast cancer, microsatellite instability highcolorectal cancer, gastric carcinoma, mesothelioma, pancreatic cancer,and cervical cancer.

In some embodiments, the tumor cells are, and/or the subject is, naïveto immunooncology therapy. Immunooncology uses the subject's immunesystem to help fight cancer. For example, an immunooncology therapyincludes, but is not limited to, atezolizumab (human monoclonal antibodythat targets PD-L1), avelumab (human monoclonal antibody that targetsPD-L1), brentuximab vedotin (antibody-drug conjugate that targets CD30),durvalamab (human monoclonal antibody that targets PD-L1), ipilimumab(human monoclonal antibody that targets CTLA-4), nivolumab (humanmonoclonal antibody that targets PD-L1), pembrolizumab (also referred toas lambrolizumab, human monoclonal antibody that targets PD-L1),tremelimumab (human monoclonal antibody that targets CTLA-4), CT-011(antibody that targets PD-1), MDX-1106 (antibody that targets PD-1),MK-3475 (antibody that targets PD-1), YW243.55.S70 (antibody thattargets PD-L1), MPDL3280A (antibody that targets PD-L1), MDX-1105(antibody that targets PD-L1), and MED14736 (antibody that targetsPD-L1). In some embodiments, the immunooncology therapy is selected froman anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody,an anti-PD-L2 antibody, an anti-TIGIT antibody (e.g., antibodiesdisclosed in WO 2015/009856).

In other embodiments, the tumor cells are, and/or the subject isresponsive to immune checkpoint therapy. In some embodiments, the cancerhas shown response to anti-PD1 therapy. For example, the cancer mayinclude non-small cell lung cancer (NSCLC), melanoma, renal cell cancer(RCC), cancer of the bladder, Hodgkin's lymphoma, and head and necksquamous cell carcinoma.

Other embodiments of the present disclosure provide a method oftreatment of infection.

Still other embodiments of the present disclosure provide a method oftreatment of infection comprising administration of a therapeuticallyeffective amount of a composition comprising an anhydrous crystallineform of the compound of formula (I) to the subject in need thereof.

In certain embodiments, the present disclosure provides uses of ananhydrous crystalline form of the compound of formula (I) for thepreparation of a medicament for the treatment of infectious disease, aswell as methods of administering a therapeutically effective amount of acomposition comprising an anhydrous crystalline form of the compound offormula (I) for the treatment of infectious disease.

In some embodiments, the infectious disease is bacterial infection,viral infection, fungal infection, or parasitic infection, as well asmethods of administering a therapeutically effective amount of acomposition comprising an anhydrous crystalline form of the compound offormula (I) for the treatment of bacterial infection, viral infection,fungal infection, or parasitic infection.

In some embodiments, for example, bacterial infection may be caused byat least one bacterium selected from anthrax, Bacilli, Bordetella,Borrella, botulism, Brucella, Burkholderia, Campylobacter, Chlamydia,cholera, Clostridium, Conococcus, Corynebacterium, diptheria.Enterubacter, Enterococcus, Erwinia, Echerichia, Francisella,Haenophilus, Heliobacter, Klebsiella, legionella, Leptospira,leptospirosis, Listeria, Lyme's disease, meningococcus, Mycobacterium,Alycoplasma, Neisseria, Pasteurelia, Pelbacter, plague, Pneumonococcus,Proteus, Pseudomonas, Rickettsia, Salmonella. Serratia, Shigella,Staphylococcus, Streptococcus, tetanus, Treponema, Vibrio, Yersnia andXanthomonas.

In other embodiments, for example, viral infection may be caused by atleast one virus selected from Adenoviridae, Papillomaviridae,Polyomaviridae, Herpesviridae, Poxviridae, H-epadnaviridae,Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae,Coronaviridae, Flaviviridae, Retroviridae, Ibgaviridae, Arenaviridae,Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae,Rhabdoviridae, and Reoviridae. In certain embodiments, the virus may bearboviral encephalitis virus, adenovirus, herpes simplex type I, herpessimplex type 2, Varicella-zoster virus, Epstein-barr virus,cytomegalovirus, herpesvirus type 8, papillomavirus, BK virus,coronavirus, echovirus, JC virus, smallpox, Hepatitis B, bocavirus,parvovirus B19, astrovirus, Norwalk virus, coxsackievirus, Hepatitis A,poliovirus, rhinovirus, severe acute respiratory syndrome virus,Hepatitis C, yellow fever, dengue virus, West Nile virus, rubella,Hepatitis E, human immunodeficiency virus (HIV), human T-celllymphotropic virus (HTLV), influenza, guanarito virus, Junin virus,Lassa virus. Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fevervirus, ebola virus, Marburg virus, measles virus, molluscum virus, mumpsvirus, parainfluenza, respiratory syncytial virus, humanmetapneumovirus, Hendra virus, Nipah virus, rabies, Hepatitis 1),rotavirus, orbivirus, coltivirus, vaccinia virus, and Banna virus.

In other embodiments, for example, fungal infection may be selected fromthrush, Aspergillus (fumigatus, niger, etc.), Blastomyces dermatitidis,Candida (albicans, krusei, glabrata, tropicalis, etc.), Coccidioidesimmitis, Cryptococcus (neoformans, etc.), Histoplasma capsulatum,Mucorales (mucor, absidia, rhizophus), Paracoccidioides brasiliensis,sporotrichosis, Sporothrix schenkii, zygomycosis, chromoblastomycosis,lobomycosis, mycetoma, onychonycosis, piedra pityriasis versicolor,tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea tavosa,tinea nigra, tinea pedis, otomycosis, phaeohyphomycosis, andrhinosporidiosis.

In some embodiments, for example, parasitic infection may be caused byat least one parasite selected from Acanthamoeba, Babesia microti,Balantidium coi, Enamoeba hystolytica, Giardia lamblia, Cryptosporidiummuris, Trypanosomatida gambiense, Trypanosoinatida rhodesiense,Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, Leishimaniabraziliensis, Leishmania tropica, L_eishmania donovani, Toxoplasmnagondmi, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae,Plasmodium falciparum, Pneumocystis carinii, Trichomonas vaginalis,Histomonas meleagridis, Secementea, Trichuris trichiura. Ascarislumbricoides. Enterobius vermicularis, Ancylostoia duodenale,Naegleriafowleri, Necator americanus, Nippostrongylus brasiliensis,Strongyloides stercoralis, Wuchereria bancroffi, Dracunculus medinensis,blood flukes, liver flukes, intestinal flukes, lung flukes, Schistosomamansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciolahepalica, Fasciola gigantica, Heterophyes heterophyes, and Paragoimuswestermani.

The term “subject” includes mammals (especially humans) and otheranimals, such as domestic animals (e.g., household pets including catsand dogs) and non-domestic animals (such as wildlife).

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence or frequency of the disorder or condition in the treatedsample relative to an untreated control sample, or delays the onset orreduces the severity of one or more symptoms of the disorder orcondition relative to the untreated control sample. Thus, prevention ofcancer includes, for example, reducing the number of detectablecancerous growths in a population of patients receiving a prophylactictreatment relative to an untreated control population, and/or delayingthe appearance of detectable cancerous growths in a treated populationversus an untreated control population, e.g., by a statistically and/orclinically significant amount. Prevention of an infection includes, forexample, reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

Pharmaceutical Compositions

In certain embodiments, the present disclosure provides a pharmaceuticalcomposition comprising an anhydrous crystalline form of the compound offormula (I) as disclosed herein, optionally admixed with apharmaceutically acceptable carrier or diluent.

The present disclosure also provides methods for formulating thedisclosed anhydrous crystalline forms of the compound of formula (I) forpharmaceutical administration.

The compositions and methods of the present disclosure may be utilizedto treat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, an anhydrous crystalline form of the compoundof formula (I) of the disclosure and a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are well known in the artand include, for example, aqueous solutions such as water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil, or injectable organic esters.In a preferred embodiment, when such pharmaceutical compositions are forhuman administration, particularly for invasive routes of administration(i.e., routes, such as injection or implantation, that circumventtransport or diffusion through an epithelial barrier), the aqueoussolution is pyrogen-free, or substantially pyrogen-free. The excipientscan be chosen, for example, to effect delayed release of an agent or toselectively target one or more cells, tissues or organs. Thepharmaceutical composition can be in dosage unit form such as tablet,capsule (including sprinkle capsule and gelatin capsule), granule,lyophile for reconstitution, powder, solution, syrup, suppository,injection or the like. The composition can also be present in atransdermal delivery system, e.g., a skin patch. The composition canalso be present in a solution suitable for topical administration, suchas an eye drop.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as ananhydrous crystalline form of the compound of formula (I) of thedisclosure. Such physiologically acceptable agents include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. The choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable agent, depends, for example, on the route of administrationof the composition. The preparation of pharmaceutical composition can bea self-emulsifying drug delivery system or a self-microemulsifying drugdelivery system. The pharmaceutical composition (preparation) also canbe a liposome or other polymer matrix, which can have incorporatedtherein, for example, an anhydrous crystalline form of the compound offormula (I) of the disclosure. Liposomes, for example, which comprisephospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); anally, rectally or vaginally (for example, as a pessary,cream or foam); parenterally (including intramuscularly, intravenously,subcutaneously or intrathecally as, for example, a sterile solution orsuspension); nasally; intraperitoneally; subcutaneously; transdermally(for example as a patch applied to the skin); and topically (forexample, as a cream, ointment or spray applied to the skin, or as an eyedrop). The compound may also be formulated for inhalation. In certainembodiments, a compound may be simply dissolved or suspended in sterilewater. Details of appropriate routes of administration and compositionssuitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as an anhydrouscrystalline form of the compound of formula (I) of the disclosure, withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present disclosure withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the disclosure suitable for oral administration may bein the form of capsules (including sprinkle capsules and gelatincapsules), cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), lyophile, powders, granules,or as a solution or a suspension in an aqueous or non-aqueous liquid, oras an oil-in-water or water-in-oil liquid emulsion, or as an elixir orsyrup, or as pastilles (using an inert base, such as gelatin andglycerin, or sucrose and acacia) and/or as mouth washes and the like,each containing a predetermined amount of a compound of the presentdisclosure as an active ingredient. Compositions or compounds may alsobe administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof, (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, orurethral administration may be presented as a suppository, which may beprepared by mixing one or more active compounds with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active compound.

Formulations of the pharmaceutical compositions for administration tothe mouth may be presented as a mouthwash, or an oral spray, or an oralointment.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices may be especially useful for delivery to thebladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present disclosure to the body. Suchdosage forms can be made by dissolving or dispersing the active compoundin the proper medium. Absorption enhancers can also be used to increasethe flux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this disclosure.Exemplary ophthalmic formulations are described in U.S. Publication Nos.2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat.No. 6,583,124, the contents of which are incorporated herein byreference in its entirety. If desired, liquid ophthalmic formulationshave properties similar to that of lacrimal fluids, aqueous humor orvitreous humor or are compatible with such fluids. A preferred route ofadministration is local administration (e.g., topical administration,such as eye drops, or administration via an implant).

A suppository also is contemplated as being within the scope of thisdisclosure.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this disclosure, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinaceous biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the anhydrous crystalline form of the compound offormula (I) of the disclosure. A larger total dose can be delivered bymultiple administrations of the agent. Methods to determine efficacy anddosage are known to those skilled in the art (Isselbacher et al. (1996)Harrison's Principles of Internal Medicine 13 ed., 1814-1882, hereinincorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the disclosure will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentdisclosure, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Examples Analytical Methods X-ray Powder Diffraction

X-Ray Powder Diffraction (XRPD) patterns were collected on an Empyreandiffractometer or an X′Pert3 diffractometer using CuKα radiation (45 kV,40 mA) or using a PANalytical Empyrean diffractometer.

The details of the data collection are summarized in Table 2:

TABLE 2 X-Ray Powder Diffraction Parameters Instrument PANalytical ModelEmpyrean X′ Pert3 Empyrean (Reflection (Reflection (Transmission Mode)Mode) Mode) X-Ray Cu, kα, wavelength Kα1 (Å): 1.540598, Kα2 (Å):1.544426 Kα2/Kα1 intensity ratio: 0.50 X-Ray tube setting 45 kV, 40 mADivergence slit Automatic Fixed ⅛° Fixed ½° Scan mode Continuous Scanrange (°2TH) 3°-40° Scan step time [s] 17.8   46.7   33.02   Step size(°2TH) 0.0167 0.0263 0.0167 Test Time (s) 5 min 30 s 5 min 04 s 10 min11 s

HPLC

Purity analysis was performed on an Agilent HP1100 series systemequipped with a diode array detector and using ChemStation softwarevB.04.03 using the method detailed below in Table 3.

TABLE 3 HPLC Parameters Parameter Value Type of method Reversed phasewith gradient elution Sample Preparation Diluent, acetonitrile/H₂O = 1:1Column ZIC-HILIC, 250 × 4.6 mm, 5 μm Column Temperature 30° C. InjectionVolume 10 μL Detector Wavelength, Bandwidth UV at 210 nm Flow Rate 1.0mL/min Mobile Phase A 10 mM KH₂PO₄ in H₂O Mobile Phase B acetonitrileGradient Timetable Time (min) % Mobile Phase A 0.0 80 2.0 80 20.0 6020.1 80 30.0 80

Thermogravimetric Analysis and Differential Scanning Calorimetry

Thermogravimetric analysis (TGA) data were collected using a TAQ500/Q5000 TGA from TA Instruments. Differential scanning calorimetry(DSC) was performed using a TA Q200/Q2000 DSC from TA Instruments.Method parameters are provided in Table 4 below.

TABLE 4 TGA and DSC Parameters Parameters TGA DSC Method Ramp Ramp PanPlatinum, open Aluminum plate, crimped Temperature RT-Target Temperature25° C.-Target Temperature Ramp rate 10° C./min 10° C./min Purge gas N₂N₂

Example 1: Synthesis of Form a by Crystallization from anIsopropanol/Water Mixture

Formula (I) (1.00 g) was weighed into a glass vial, and deionised water(10 mL), which afforded a clear solution after being stirred at 35° C.Isopropanol (2 mL) was added followed by formula (I) Form A seedcrystals. Charged with 18 mL isopropanol at 35° C. over 8 h, then heldfor 1 h. Cooled to 25° C. in 2 h, and held at 25° C. for ˜1 h. The whitesolid present was then isolated by filtration, and shown by XRPDanalysis to be anhydrous crystalline formula (I) Form A (FIG. 1 ). Thesolid was then dried under vacuum at 40° C. for about 20 h (yield notdetermined).

Example 2: Synthesis of Form a by Crystallization from an Ethanol/WaterMixture A. 5 wt % Seed Crystals, 8 h EtOH Addition

Formula (I) (1.02 g) was charged into a 100 mL reactor with deionisedwater (10 mL). The mixture was stirred (about 300 rpm, about 40 m/min)with an overhead stirrer at 35° C. to afford a clear solution. Thesolution was charged with 2 mL EtOH followed by 1-2 mg of seed crystals(formula (I) Form A). The seeding point was when the EtOH/H₂O ratio was1:5 v/v. The My of the seed crystals was 34.2 μm with partlyagglomerated crystals, and the D50 of the dry milled seed crystals was14.9 μm. A cloudy suspension was observed. An additional 50.0 mg of seedcrystals (formula (I) Form A) was added, and the mixture was held at 35°C. without stirring for about 10 min. The resulting slurry was stirredat 35° C. for about 8 h while 18 mL of EtOH was slowly added. Themixture was then held at 35° C. without stirring for about 1 h. Themixture was cooled to 20° C. over 1 h and held at temperature withoutstirring for about 1 h. The white solid present was then isolated byfiltration, and washed with 2×10 mL EtOH. The cake was dried at 40° C.under vacuum for 20 h to afford 0.86 g solid (˜86% solid yield). Thesolid was shown by XRPD analysis to be anhydrous crystalline formula (I)Form A (FIG. 1 ).

B. 5 wt % Seed Crystals, 4 h EtOH Addition

The procedure outlined in Example 2A was followed with 1.00 g of Formula(I) in 10 mL of water, an additional 50.1 mg of seed crystals (formula(I) Form A), and slow addition of 18 mL of EtOH over 4 h to afford 0.85g solid (˜85% solid yield). The solid was shown by XRPD analysis to beanhydrous crystalline formula (I) Form A (FIG. 2 ).

C. 1 wt % Seed Crystals, 4 h EtOH Addition

The procedure outlined in Example 2A was followed with 1.03 g of Formula(I) in 10 mL of water, an additional 10.6 mg of seed crystals (formula(I) Form A), and slow addition of 18 mL of EtOH over 4 h to afford 0.87g solid (˜87% solid yield). The solid was shown by XRPD analysis to beanhydrous crystalline formula (I) Form A (FIG. 2 ).

D. 5 wt % Seed Crystals, 4 h EtOH Addition

Formula (I) (1.01 g) was charged into a 100 mL reactor with deionisedwater (8 mL). The mixture was stirred (about 450 rpm, about 60 m/min)with an overhead stirrer at 35° C. to afford a clear solution. Thesolution was charged with 1 mL EtOH followed by 1˜2 mg of seed crystals(formula (I) Form A). The seeding point was when the EtOH/H₂O ratio was1:8 v/v. The My of the seed crystals was 34.2 μm with partlyagglomerated crystals, and the D50 of the dry milled seed crystals was14.9 μm. A cloudy suspension was observed. An additional 50.6 mg of seedcrystals (formula (I) Form A) was added, and the mixture was held at 35°C. without stirring for about 20 min. The resulting slurry was stirredat 35° C. for about 4 h while 21 mL of EtOH/H₂O (19:1 v/v) was slowlyadded. The mixture was then held at 35° C. without stirring for about 1h. The mixture was cooled to 20° C. over 1 h and held at temperaturewithout stirring for about 1 h. The white solid present was thenisolated by filtration, and washed with 2×10 mL EtOH. The cake was driedat 40° C. under vacuum for 7 h to afford 0.87 g solid (˜87% solidyield). The solid was shown by XRPD analysis to be anhydrous crystallineformula (I) Form A (FIG. 2 ).

E. Milled Seed Crystals

A 100 mL V style reactor was charged with formula (I) Form A (1.2 g) andheld at 10° C. with magnetic stirring using a cross stir bar during theprocedure. The reactor was charged with 20 mL of EtOH with a sonicationprobe. The mixture was sonicated for about 100 min. The white solidpresent was then isolated by filtration, and washed with 2×10 mL EtOH.The cake was dried at room temperature under vacuum for 10 h.

Agglomerates of rod-like crystals (formula (I) Form A) were obtained inall experiments, with My ranging from 76.2 to 97.6 μm and loose densityof 0.13˜0.16 g/mL (tap density: 0.19-0.30 g/mL). No significantdifference was observed in morphology, particle size distribution (PSD),and loose density among these three experiments (Examples 2A-2C). Themorphology and particle size of the products is consistent before andafter drying. Not wishing to be bound by any particular theory,agglomerates may be caused by high local supersaturation at the entrypoint of anti-solvent. Another experiment was performed using EtOH/H₂O(19:2, v/v) as anti-solvent, with an aim to reduce local supersaturation(Example 2D). Agglomeration was mitigated compared with experiments withEtOH as anti-solvent. My of resulting crystals was 63.1 μm and loosedensity was 0.16 g/mL. Tap density jumped to 0.38 g/mL. Rod-likecrystals were observed in the EtOH/water systems using polarized lightmicroscopy of the wet cake and the dried cake (FIGS. 3A-6B). Theparticle size distribution of anhydrous crystal Form A of formula (I)based on the various crystallization conditions is summarized in Table5. The particle size is consistent before and after drying withsonication at 30 W for 30 s of the sample. There are differences in theparticle size before and after sonication (e.g., 4C dry before v. 4C dryafter).

TABLE 5 Particle Size Distribution of Anhydrous Crystal Form A ofFormula (I) Mv D10 D50 D90 Loose d Tap d Solvent Ex. (μm) SD (μm) (μm)(μm) (g/mL) (g/mL) EtOH 4A wet 76.3 39.0 26.2 68.3 132.1 0.15 0.29 4Adry 76.2 39.1 25.7 68.2 131.9 0.15 0.29 4B wet 80.7 39.6 29.5 74.2 136.10.16 0.30 4B dry 81.1 40.9 27.3 73.8 138.4 0.16 0.30 4C dry 168.7 53.097.4 167.4 244.5 before 4C dry 97.6 62.2 24.1 98.2 171.4 0.13 0.26 afterEtOH/ 4D dry 76.1 31.1 34.4 70.8 120.8 H₂O before (19:2, 4D dry 63.129.8 22.7 58.3 104.0 0.16 0.38 v/v) after Milled 4E dry 145.2 164.1 6.4356.6 391.5 Seed before 4E dry 34.2 19.2 3.1 14.9 69.9 after

F. Seed Crystals, EtOH Addition

Formula (I) (1.02 g) was weighed into a glass vial, and deionised water(10 mL) was added, which afforded a clear solution after being stirredat 35° C. EtOH (2 mL) was added, followed by formula (I) Form A seedcrystals. Charged 18 mL EtOH at 35° C., then held for 1 h. Cooled to 20°C., held for at 20° C.˜1 h. The white solid present was then isolated byfiltration, and was shown by XRPD analysis to be formula (I) anhydrousForm A. The solid was then dried under vacuum at RT for about 22 h (0.86g, ˜80% recovery).

G. Seed Crystals, 19:2 EtOH/H₂O Addition

Formula (I) (1.01 g) was weighed into a glass vial, and deionised water(8 mL) was added which afforded a clear solution after being stirred at35° C. EtOH (1 mL) was added followed by formula (I) Form A seedcrystals. Charged the mixture with 21 mL EtOH/H₂O (19:2, v/v), then heldfor ˜1 h. Cooled to 20° C., and held at 20° C. for ˜1 h. The white solidpresent was then isolated by filtration, and was shown by XRPD analysisto be formula (I) anhydrous Form A. The solid was then dried undervacuum at 40° C. for about 7 h (0.87 g, 82% recovery).

H. Seed Crystals, 5:1 EtOH/H₂O Addition

Formula (I) (1.01 g) was weighed into a glass vial, and deionised water(8 mL) was added which afforded a clear solution after being stirred at35° C. EtOH (1 mL) was added followed by formula (I) Form A seedcrystals. Charged the mixture with 30 mL EtOH/H₂O (5:1, v/v), then heldfor ˜1 h. Cooled to 20° C., and held at 20° C. for ˜1 h. The white solidpresent was then isolated by filtration, and was shown by XRPD analysisto be formula (T) anhydrous Form A. The solid was then dried undervacuum at 40° C. for about 6 h (0.84 g, ˜80% recovery).

I. 5 wt % Seed Crystals, 8 h EtOH Addition

Formula (I) (1.02 g) was weighed into a 100 mL reactor with the additionof 10 mL deionised water. After being stirred for 10 min to get a clearsolution, 2 mL EtOH solvent was added, then 1˜2 mg formula (I) Form Aseeds were added into the reactor. A muddy or cloudy phenomenon wasobserved. The solution was kept up stirring for 10 min followed byadding ˜50 mg formula (I) Form A seeds. Afterwards the solution wasstirred for about 20 min. Then, 18 mL EtOH solvent was added byinjection pump over eight hours at 35° C. Then the turbid solution waskept for one hour at 35° C. Lastly, the turbid solution was cooled to20° C. using one hour, which was then kept at 20° C. for about 11 hours.

Example 3: Synthesis of Form a by Slurry A. 1:9 EtOH/H₂O

Formula (I) (340.8 mg) was weighed into a glass vial. 1 mL of solvent(water/EtOH=9/1, v/v) was added. The resulting slurry was stirred at 50°C. for about 15 h. The wet solid present was then isolated bycentrifugation, and was shown by XRPD analysis to be formula (I)anhydrous Form A (yield not determined).

B. 2:1 EtOH/H₂O

Formula (I) (29.4 mg) was weighed into a glass vial. 1 mL of solvent(water/EtOH=1/2, v/v) was added. The resulting slurry was stirred at 50°C. for about 15 h. The wet solid present was then isolated bycentrifugation, and was shown by XRPD analysis to be formula (I)anhydrous Form A (yield not determined).

C. 1:9 IPA/H₂O

Formula (I) (327.3 mg) was weighed into a glass vial. 1 mL of solvent(water/isopropanol=9/1, v/v) was added. The resulting slurry was stirredat 50° C. for about 15 h. The wet solid present was then isolated bycentrifugation, and was shown by XRPD analysis to be formula (I)anhydrous Form A (yield not determined)

D. 2:1 IPA/H₂O

Formula (I) (29.8 mg) was weighed into a glass vial. 1 mL of solvent(water/isopropanol=1/2, v/v) was added. The resulting slurry was stirredat 50° C. for about 15 h. The white solid present was then isolated bycentrifugation, and was shown by XRPD analysis to be formula (I)anhydrous Form A (yield not determined).

Example 4: Thermogravimetric Analysis and Differential ScanningCalorimetry of Formula (I)

Thermogravimetric analysis (TGA) of formula (I) anhydrous Form A showeda weight loss of 0.5% up to 150° C., which is consistent with ananhydrate form. Differential scanning calorimetry (DSC) showed anendotherm at 203.5° C. (onset temperature) due to melting/decomposition(FIG. 7 ).

About 40 mg of a sample of formula (I) Form A was kept at eachcorresponding condition for one month without significant changes. Ashoulder peak was observed in DSC curves of the initial sample andsamples after storage at 25° C./60% Relative Humidity (RH) and 40°C./75% RH for one month or for three months. No significant effect ofthe shoulder peaks on form or HPLC purity was observed. See Table 6.

TABLE 6 Solid Stability of Anhydrous Crystal Form A of Formula (I) DSC(° C., onset) Final TGA Melting Shoulder Condition Form (%) Peak PeakInitial Form A 0.40 200.6 193.7 25° C., 60% RH, 1 month Form A 0.60200.7 192.9 25° C., 60% RH, 3 months Form A 2.8 201.4 194.3 40° C., 75%RH, 1 month Form A 1.0 201.1 193.4 40° C., 75% RH, 3 months Form A 1.9200.9 194.1 60° C., sealed, 1 month Form A 0.6 201.0 No 60° C., sealed,3 months Form A 2.1 200.2 No Seed (after ground), Form A 0.4 199.9 No 5°C., sealed

Example 5: Stability and Forced Degradation Study for AnhydrousCrystalline Formula (I)

Solid Stability

Samples of formula (I) anhydrous crystalline Form A were stored assolids at 25° C./60% RH, 30° C./-56%, and 40° C./75% RH for the timeperiods listed below. The samples were prepared in duplicate with anoffset of 2 weeks. Each replicate was stored in a different container.

Formula (I) anhydrous crystalline Form A remained unchanged in terms ofsolid form and particle morphology after storage for one month and threemonths at 25° C./60% RH, 40° C./75% RH, and 60° C. See FIG. 8 after 3months. The chemical purity of a solid sample of about 40 mg was alsoevaluated, and no significant degradation was observed after storage forone month. See Table 7.

TABLE 7 HPLC Purity Profiles of Solid Stability of Anhydrous CrystalForm A of Formula (I) Initial 25° C., 40° C., 60° C., Peak RRT (area %)60% RH 75% RH sealed 1 month Purity (area %) Impurity 1 0.63 0.63 <0.05<0.05 <0.05 Impurity 2 0.75 <0.05 0.07 0.08 0.07 Impurity 3 0.79 0.070.11 0.11 0.08 Form A 1.00 99.86 99.83 99.81 99.85 3 months Purity (area%) Impurity 1 0.63 0.63 <0.05 <0.05 <0.05 Impurity 2 0.75 <0.05 0.100.12 0.11 Impurity 3 0.79 0.07 0.10 0.09 0.09 Form A 1.00 99.86 99.8099.79 99.74

Solution Stability

Samples of formula (I) anhydrous crystalline Form A were stored assolutions in water mixed with acetonitrile, tetrahydrofuran, isopropylalcohol, or ethanol at room temperature and 35° C. for 7-8 days. Thesample concentration was about 10 mg/mL and was kept stirring.

At room temperature, a drop in chemical purity was observed from about99.6% (time 0) to about 99.1% to 99.2% (time 24 h). The main growingimpurity observed by HPLC eluted at RRT 0.72 (about 0.36% to about 0.40%at the 24 h time point). (See Table 8.)

TABLE 8 HPLC Purity Profiles of Solution Stability of Formula (I)Anhydrous Crystal Form A at RT Imp 1 Imp 2 Imp 3 Imp 4 Imp 5 Imp 6 APITime (RRT (RRT (RRT (RRT (RRT (RRT (RRT Solvent (h) 0.42) 0.53) 0.72)0.81) 0.82) 0.94) 1.00) ACN/H₂O 0 0.05 <0.05 0.05 <0.05 0.25 <0.05 99.65(1:1, v/v) 4 0.05 <0.05 0.12 0.07 0.20 0.06 99.51 24 0.05 0.05 0.36<0.05 0.20 0.10 99.24 THF/H₂O 0 <0.05 <0.05 0.05 <0.05 0.26 <0.05 99.64(1:1, v/v) 4 0.05 <0.05 0.15 <0.05 0.26 0.06 99.47 24 0.05 <0.05 0.38<0.05 0.23 0.10 99.19 IPA/H₂O 0 0.05 <0.05 0.06 <0.05 0.25 0.05 99.59(1:2, v/v) 4 <0.05 <0.05 0.15 0.06 0.22 0.06 99.52 24 0.05 0.05 0.38<0.05 0.22 0.08 99.21 EtOH/H₂O 0 0.05 <0.05 0.08 <0.05 0.24 0.05 99.58(1:1, v/v) 4 0.05 <0.05 0.16 <0.05 0.26 0.06 99.47 24 0.05 0.08 0.40<0.05 0.22 0.14 99.11

At 35° C., a drop in chemical purity was observed from about 99.6% (time0) to about 98.200 to 98.400 (time 24 h). The main growing impurityobserved by HIPLC eluted at RRT 0.72 (about 1.100% to about 1.3200 atthe 24 h time point). (See Table 9.)

TABLE 9 HPLC Purity Profiles of Solution Stability of Formula (I)Anhydrous Crystal Form A at 35° C. Imp 1 Imp 2 Imp 3 Imp 4 Imp 5 Imp 6API Time (RRT (RRT (RRT (RRT (RRT (RRT (RRT Solvent (h) 0.42) 0.53)0.72) 0.81) 0.82) 0.94) 1.00) ACN/H₂O 0 0.05 <0.05 0.08 <0.05 0.25 0.0599.58 (1:1, v/v) 4 <0.05 <0.05 0.21 <0.05 0.20 0.05 99.44 24 0.09 <0.051.10 0.06 0.17 0.15 98.42 THF/H₂O 0 <0.05 <0.05 0.08 <0.05 0.27 0.0699.56 (1:1, v/v) 4 0.05 <0.05 0.25 0.07 0.21 0.06 99.37 24 0.08 <0.051.24 0.05 0.17 0.16 98.30 IPA/H₂O 0 <0.05 <0.05 0.10 <0.05 0.24 0.0599.57 (1:2, v/v) 4 0.05 <0.05 0.25 0.07 0.20 0.08 99.35 24 0.05 0.050.38 <0.05 0.22 0.08 99.21 EtOH/H₂O 0 0.05 <0.05 0.08 <0.05 0.24 0.0599.58 (1:1, v/v) 4 0.05 <0.05 0.16 <0.05 0.26 0.06 99.47 24 0.05 0.080.40 <0.05 0.22 0.14 99.11

Example 6: Solubility Measurement for Anhydrous Crystalline Formula (I)

Formula (I) anhydrous Form A showed good solubility in simulated gastricfluid (SGF) of greater than 114.0 mg/mL at room temperature. In a watersolution with pH adjustments by NaOH and HCl solutions at roomtemperature, the solubility of formula (I) Form A was 87.5<S<116.7 at pH7.08 and 83.5<S<111.3 mg/mL at pH 9.14. The pH values of the aqueoussolutions after the solubility test were 5.13 and 5.15, respectively.

Equilibrium solubility of formula (I) anhydrous Form A was measured inthe water at 20° C. and 30° C. All samples were equilibrated attemperature for 6 hrs, and the solubility of supernatant was measured byHPLC, while the solids were checked by XRPD. (See Table 10.)

TABLE 10 Equilibrium Solubility of Anhydrous Crystal Form A of Formula(I) Solubility Starting Form Solvent Temp. (° C.) Final Form (mg/mL)Form A Water 20 Form A 125.0 Form A 30 Form A 137.2

No form change was observed during the solubility testing for Form A(FIG. 9 ).

Example 7: Dynamic Vapor Sorption for Anhydrous Crystalline Formula (I)

As the dynamic vapor sorption (DVS) results showed, the masscontinuously increased with increasing humidity for formula (I) Form A(FIG. 10 ). Anhydrous Form A also showed about 0.66% mass change up to80% RH, which indicated that the sample was slightly hygroscopic. Noform change was observed during the DVS testing (FIG. 11 ).

Example 8: Single Crystal Structure Determination

Form A

Form A was crystallized from an ethanol/water mixture as described inExample 21. The crystals had a rod-like shape (FIG. 3A). The singlecrystal X-ray diffraction data was collected at 296 K using Bruker® D8VENTURE diffractometer (Mo/Kα radiation; λ=0.71073 Å). The structuralinformation and refinement parameters are given in Table 11.

TABLE 11 Structural Information and Refinement Parameters for CrystalForm A of Formula (I) Empirical formula C₁₂H₂₀N₆O₇ Temperature 296 KWavelength Mo/Kα (λ = 0.71073 Å) Crystal system, space group Monoclinic,P2₁ Unit cell dimensions a = 10.197(6) Å α = 90° b = 6.815(4) Å β =106.355(12)° c = 11.676(5) Å γ = 90° Volume 778.6(7) Å³ Z, Calculateddensity 2, 1.537 g/cm³ Absorption coefficient 0.127 mm⁻¹ F(000) 380.0Crystal size 0.08 × 0.04 × 0.02 mm³ 2 Theta range for data collection6.252 to 55.072° Limiting indices −13 ≤ h ≤ 8 −8 ≤ k ≤ 6 −12 ≤ l ≤ 13Reflections collected/ 4208/2897 [R(int) = 0.0424] Independentreflections Completeness 80.67% Refinement method Full-matrixleast-squares on F² Data/restraints/parameters 2897/1/230Goodness-of-fit on F² 1.022 Final R indices [I > 2sigma(I)] R₁ = 0.0568,wR₂ = 0.0918 Largest diff. peak and hole 0.24/−0.28 e · Å⁻³ Flackparameter −0.1(10)

Single crystal structural analysis confirmed that the crystallineformula (I) Form A is an anhydrate with the asymmetric unit comprised ofone formula (I) molecule (FIG. 12A). The bond lengths of the C—O/C═Ofrom the carboxyl group were similar (C—O/C═O: 1.256 Å/1.241 Å). Therewere three residual electron density peaks (0.35, 0.31, and 0.25 e·Å⁻³)assigned as the hydrogen atoms around the N1 atom at the distances of1.031 Å, 0.887 Å, and 0.940 Å, respectively. Therefore, it is suggestedthat the formula (I) molecule was a zwitterion in the anhydrous Form Acrystal (FIGS. 12B and 12C). In anhydrous crystalline Form A, adjacentmolecules of the compound of formula (I) connect to each other to formthe 3-D packing structure via the hydrogen bonds (O—H—O, N—H—O).

The absolute configuration of the formula (I) molecule could not bedetermined with the diffraction data due to the weak anomalousscattering behavior of the formula (I) molecule. The experimental XRPDis compared to the calculated XRPD pattern based on the single crystalstructure showing consistent peaks (FIG. 13 ).

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1-43. (canceled)
 44. A method of treating cancer in a subject in needthereof comprising administering to the subject an anhydrous crystallinecompound having the structure of formula (I),


45. The method of claim 44, wherein the anhydrous crystalline compoundhas 2θ values 18.8±0.2, 20.5±0.2, 26.4±0.2, 27.5±0.2, and 32.0±0.2. 46.The method of claim 44, wherein the anhydrous crystalline compound has2θ values 18.8±0.2, 20.5±0.2, 26.1±0.2, 26.4±0.2, 27.5±0.2, 30.6±0.2,and 32.0±0.2.
 47. The method of claim 44, wherein the anhydrouscrystalline compound has 2θ values 15.8±0.2, 16.5±0.2, 18.8±0.2,20.5±0.2, 25.5±0.2, 26.1±0.2, 26.4±0.2, 27.5±0.2, 30.6±0.2, and32.0±0.2.
 48. The method of claim 44, wherein the anhydrous crystallinecompound has 2θ values 15.8±0.2, 16.5±0.2, 18.8±0.2, 20.5±0.2, 21.8±0.2,22.3±0.2, 24.2±0.2, 25.5±0.2, 26.1±0.2, 26.4±0.2, 27.5±0.2, 30.4±0.2,30.6±0.2, and 32.0±0.2.
 49. The method of claim 44, wherein theanhydrous crystalline compound has 2θ values 10.2±0.2, 15.2±0.2,15.8±0.2, 16.5±0.2, 17.6±0.2, 18.1±0.2, 18.8±0.2, 20.5±0.2, 21.8±0.2,22.3±0.2, 22.9±0.2, 24.2±0.2, 25.4±0.2, 26.1±0.2, 26.4±0.2, 27.5±0.2,28.1±0.2, 28.8±0.2, 29.3±0.2, 30.4±0.2, 30.6±0.2, 32.0±0.2, 33.4±0.2,33.6±0.2, 34.6±0.2, 35.8±0.2, 36.7±0.2, 37.3±0.2, 38.2±0.2, 38.6±0.2,and 39.2±0.2.
 50. The method of claim 44, wherein the cancer is selectedfrom blastoma, breast cancer, epithelial cancer, colon cancer, lungcancer, melanoma, prostate cancer, renal cancer, bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, uterine cancer, ovariancancer, colorectal cancer, rectal cancer, cancer of the anal region,cancer of the peritoneum, stomach cancer, testicular cancer, carcinomaof the fallopian tubes, carcinoma of the endometrium, cervical cancer,vaginal cancer, vulvar cancer, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, thyroid cancer, cancerof the parathyroid gland, cancer of the adrenal gland, sarcoma, cancerof the urethra, cancer of the penis, chronic leukemia, acute leukemia,solid tumors of childhood, Hodgkin's lymphoma (HL), nodular sclerosis(NSHL), mixed cellularity (MCHL) and lymphocyte depleted (LDHL)), B-celllymphomas, non-Hodgkin's lymphoma, AIDS-related lymphoma, cutaneousT-cell lymphoma, Waldenstrom's macroglobulinemia, post-transplantlymphoproliferative disorder (PTLD), lymphocytic lymphoma, primary CNSlymphoma, T-cell lymphoma, mesothelioma, thymic carcinoma, myeloma,cancer of the bladder, cancer of the ureter, carcinoma of the renalpelvis, and liver cancer.
 51. The method of claim 44, wherein the canceris selected from small cell lung cancer, multiple myeloma, bladdercarcinoma, primary ductal carcinoma, ovarian carcinoma, Hodgkin'slymphoma, gastric carcinoma, acute myeloid leukemia, and pancreaticcancer.
 52. The method of claim 44, wherein the cancer is selected fromcarcinoma of the endometrium, ovarian cancer, Hodgkin's lymphoma,non-Hodgkin's lymphoma, and leukemia.
 53. The method of claim 44,wherein the cancer is selected from prostate adenocarcinoma, lungadenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma,breast cancer and colorectal adenocarcinoma.